Methods for treating allograft products

ABSTRACT

There are disclosed systems and methods of delivering sterile fluids aseptically to a sealed canister. In an embodiment, a includes a canister having an inlet, a vent, and a drain outlet, a reagent manifold in communication with the inlet, a bubbler in communication with the vent, and a fluid communicator from the drain outlet. In another embodiment, a method includes providing a canister having an inlet, a vent, and a drain outlet; selectively providing reagents to the canister from a reagent manifold in communication with the inlet, allowing excess gasses to leave the canister with a bubbler in communication with the vent, and selectively purging the reagents from the canister through the drain outlet. Other embodiments are also disclosed.

REFERENCE TO PENDING PRIOR PATENT APPLICATION

This is a divisional of prior U.S. patent application Ser. No.12/454,759, now U.S. Pat. No. 7,776,291 B2 filed May 22, 2009 by RaymondJ. Klein et al. for APPARATUS FOR TREATING ALLOGRAFT PRODUCTS, which inturn is a continuation-in-part of U.S. patent application Ser. No.12/034,150, filed Feb. 20, 2008 now U.S. Pat. No. 7,794,653 by Chad J.Ronholdt, et al., for APPARATUS AND METHODS FOR TREATING ALLOGRAFTPRODUCTS, which in turn is a divisional of U.S. patent application Ser.No. 11/557,393, filed Nov. 7, 2006 now U.S. Pat. No. 7,658,888 by ChadJ. Ronholdt, et al., for APPARATUS AND METHODS FOR TREATING ALLOGRAFTPRODUCTS, which in turn claims the benefit under 35 U.S.C. 119(e) ofU.S. Provisional Patent Application Ser. No. 60/757,914, filed Jan. 10,2006, by Chad J. Ronholdt, et al., for APPARATUS AND METHODS FORTREATING ALLOGRAFT PRODUCTS. The above-identified patent applicationsare hereby incorporated herein by reference.

BACKGROUND

The use of musculoskeletal allograft tissue in reconstructive orthopedicprocedures and other medical procedures has markedly increased over thelast decade. Over the past decade, more than five millionmusculoskeletal allografts have been safely transplanted. The mostcommon allograft is bone. However, tendons, skin, heart valves andcorneas are other common types of tissue allografts.

Prior to use, the allograft tissue must be treated with various agentsin order to substantially eliminate microbial contamination as well asclean the tissue of residual blood constituents, bone marrow, residualconnective tissue and gross musculature. A variety of cleaning processeshave been developed in order to remove contaminants from the allograftand to inactivate microbial contaminants remaining on the allografts.However, these cleaning and inactivation methods are laborious andtedious, and often do not provide a high level of assurance that theallografts have been sufficiently cleaned (e.g., low or inconsistent logreductions in microbial contamination). In particular, many existingallograft cleaning processes require considerable manipulation of theallografts between steps, thus increasing the possibility ofenvironmental cross-contamination. Existing processes also tend to behard to regulate and control, and their efficacy can be techniciandependent. Existing processes also tend to have a shielding or layeringeffect that can greatly reduce ultrasonic energy penetration and thusnot clean as effectively. Furthermore, the shielding effect will alsoimpede the liberation of contaminant microorganisms off of the tissuesand into solution where they are more readily eradicated.

Following treatment, allograft products must be tested for bacterialcontamination prior to release of the tissue for transplantation.Existing methods of assessing microbial contamination, however, sufferform the same limitations described above (e.g. considerablemanipulation between steps, possibility for environmentalcross-contamination, hard to regulate and control, technician dependent,etc.).

In the past, ultrasound has been utilized to reduce and/or eliminatemicrobial contamination of allograft products. Ultrasound ismicrobiostatic to most microbes, and is used primarily to reducemicrobial loads from inanimate objects with specific bactericidalactivity on gram-negative bacteria.

With the increased use of allograft products, there is a need to provideimproved methods and apparatus for treating allografts in order to helpprovide the cleanest and safest allografts as well as confirm that theallografts are free from bacterial contamination.

SUMMARY OF THE INVENTION

The present invention provides an apparatus for treating allografts,comprising a sonication tank configured to transmit ultrasonic energy tothe interior of the tank, a treatment canister rotatably positioned inthe sonication tank, and configured to receive allografts therein, and atreatment fluid source in fluid communication with the treatmentcanister. In one embodiment, the treatment canister may be foraminoussuch that fluid within the sonication tank will pass into the treatmentcanister. Alternatively, the sonication tank may contain a sonicationfluid and the treatment canister is sealed with respect to thesonication tank such that the sonication fluid cannot enter the interiorof the treatment canister. A plurality of treatment fluid sources may beprovided in selective fluid communication with the interior of thecleaning canister, and a fluid control system adapted for exchangingtreatment fluid in the treatment canister without removing theallografts from the treatment canister may also be included.

In one embodiment, the apparatus may include at least first and secondtreatment fluid sources, with the control system adapted for deliveringthe first treatment fluid to the treatment canister and for replacingthe first treatment fluid in the treatment canister with the secondtreatment fluid. A treatment fluid outlet in fluid communication withthe treatment canister may also be included, along with a filter inselective fluid communication with the treatment fluid outlet. Theinterior of the treatment canister may also have a non-circularcross-sectional shape in a plane orthogonal to the rotational axis.

A method of treating allografts is also provided, and includes the stepsof: providing an ultrasonic treatment apparatus, the treatment apparatusincluding a sonication tank, a treatment canister rotatably mountable inthe sonication tank, and at least one ultrasonic transducer configuredto transmit ultrasonic energy to the interior of the tank and thetreatment canister; placing one or more allografts inside the treatmentcanister; and exposing the allografts to at least one treatment fluid inthe treatment canister while applying ultrasonic energy to theallografts and rotating the treatment canister in the sonication tank.In one embodiment, ultrasonic energy is applied to the allografts at afrequency between about 40 kHz and about 170 kHz with a power output ofbetween about 100 watts/gallon and about 550 watts/gallon. In anotherembodiment, ultrasonic energy is applied to the allografts at afrequency between about 72 kHz and about 104 kHz with a power output ofbetween about 100 watts/gallon and about 300 watts/gallon. In oneembodiment, the allografts are sonicated at a temperature of betweenabout 20.degree. C. and about 50.degree. C. (i.e., the temperature ofthe sonication fluid). Alternatively, the allografts are sonicated at atemperature of between about 45.degree. C. and about 50.degree. C.

The step of exposing the allografts to at least one treatment fluid maycomprise providing a treatment fluid within the treatment canister, andfurther comprise the step of exchanging at least a portion of thetreatment fluid in the treatment canister without removing theallografts from the treatment canister. The allografts may be exposed tofirst and second treatment fluids, wherein the first treatment fluid isinitially provided in the treatment canister and is thereafter exchangedfor the second treatment fluid in the treatment canister. In oneembodiment, the first and second treatment fluids are chosen from thegroup consisting of: detergents, enzyme solutions, antibiotic solutions,oxidizing agents, alcohols, sterile water, and mixtures of theforegoing. The treatment method may also include the steps of: providingan extraction fluid in the treatment canister; applying ultrasonicenergy to the allografts while rotating the treatment canister; andanalyzing the extraction fluid for microbial contamination.

In an embodiment, there is provided a system for delivering sterilefluids aseptically to a sealed canister, the system comprising acanister having an inlet, a vent, and a drain outlet, a reagent manifoldin sealed fluid communication with the inlet of the canister, whereinthe reagent manifold selectively provides a selected one of a pluralityof reagents to the canister; a bubbler in sealed fluid communicationwith the vent of the canister, wherein the bubbler allows excess gassesto leave the canister; and a fluid communicator from the drain outlet toa drain, wherein the drain outlet selectively purges the selected one ofthe plurality of reagents from the canister.

In another embodiment, there is provided a method of delivering sterilefluids aseptically to a sealed canister, the method comprising providinga canister having an inlet, a vent, and a drain outlet; selectivelyproviding a selected one of a plurality of reagents to the canister froma reagent manifold in sealed fluid communication with the inlet of thecanister; allowing excess gasses to leave the canister with a bubbler insealed fluid communication with the vent of the canister; andselectively purging the selected one of the plurality of reagents fromthe canister through the drain outlet into a fluid communicator to adrain.

Other embodiments are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention are illustrated in thedrawings, in which:

FIG. 1 is a schematic illustration of a treatment system according toone embodiment of the present invention;

FIG. 2 is a schematic illustration of a treatment system according toanother embodiment of the present invention;

FIG. 3 is a schematic illustration of a treatment canister according toone embodiment of the present invention;

FIG. 4 is a schematic illustration of a treatment canister according toanother embodiment of the present invention;

FIG. 5 is a schematic illustration of a treatment system according toanother embodiment of the present invention;

FIG. 6 is a schematic illustration of a treatment system according toyet another embodiment of the present invention;

FIG. 7 is a schematic illustration of a treatment system according toone embodiment of the present invention;

FIG. 8 illustrates a portion of a system for delivering sterile fluidsaseptically to a sealed canister;

FIG. 9 illustrates another portion of the system for delivering sterilefluids aseptically to a sealed canister shown in FIG. 8;

FIG. 10 illustrates a bubbler component of the system for deliveringsterile fluids aseptically to a sealed canister shown in FIGS. 8 and 9;

FIG. 11 is a schematic illustration of one of the canisters of thesystem for delivering sterile fluids aseptically to a sealed canistershown in FIGS. 8 and 9; and

FIGS. 12A-12D illustrate various views of one of the canisters of thesystem for delivering sterile fluids aseptically to a sealed canistershown in FIGS. 8 and 9.

The embodiments set forth in the drawing are illustrative in nature andare not intended to be limiting of the invention defined by the claims.Moreover, individual features of the drawings and the invention will bemore fully apparent and understood in view of the detailed description.

DETAILED DESCRIPTION

The present invention provides apparatus and methods for treatingallografts. As used herein, the terms “treating” and “treatment” areintended to encompass both the cleaning of allografts and theinactivation of microbial contaminants present on allografts (includinginactivation of microbial contaminants after such contaminants have beencleaned from the allografts). The apparatus and methods of the presentinvention also facilitate determining microbial contamination ofallograft products following treatment.

The apparatus and methods of the present invention provide treatment ofallografts wherein the allografts are sonicated in a treatment canisterwhile the allografts are rotating. Applicants have discovered that byrotating the allografts within the ultrasonic field, treatment issignificantly improved. While not being limited to a theory, applicantsbelieve that rotation prevents shielding of the ultrasonic energy sincethe allografts will not remain stacked on top of each other, therebyhelping to ensure that all of the allografts receive maximum exposure tothe ultrasonic energy and facilitates chemical reagent penetration.

During sonication and rotation, the allografts may be immersed in avariety of treatment solutions. In some embodiments, the treatmentprocess involves sonication of the allografts in a plurality of varioustreatment fluids in a step-wise fashion. In addition, some embodimentsof the present invention allow for various types of treatment fluids tobe supplied to, and thereafter removed from, the treatment canisterwithout opening the treatment canister. In this fashion, the allograftsare not exposed to the environment or any technician manipulation duringthe treatment process, thereby reducing the possibility ofcross-contamination. The treatment process may even be automated (orsemi-automated), thereby providing greater control over the process andmore consistent results which are less technician dependent.

FIG. 1 is a schematic illustration of one exemplary embodiment of atreatment apparatus according to the present invention. In particular,ultrasonic treatment apparatus 20 comprises a sonication tank 21 and atreatment canister 40 rotatably mounted in sonication tank 21. In theembodiment shown in FIG. 1, treatment canister 40 is sealed with respectto the interior of sonication tank 21 and is configured to receive oneor more allografts therein. As further described herein, however,treatment canister 40 may be foraminous such that fluid withinsonication tank 21 will pass into the interior of treatment canister 40.

In the embodiment of FIG. 1 wherein treatment canister 40 is sealed withrespect to the interior of sonication tank 21, a sonication fluid 22 isprovided within sonication tank 21. In the embodiment shown, asufficient amount of sonication fluid 22 is provided such that treatmentcanister 40 is completely or at least half way submerged. One or moreultrasonic transducers 30 are also provided, and may be located, forexample, in the interior of the walls of sonication tank 21. Ultrasonictransducers 30 are configured to transmit ultrasonic energy into theinterior of sonication tank 21, particularly into sonication fluid 22.Such ultrasonic energy will then be transmitted through the wall oftreatment canister 40 such that allografts contained within treatmentcanister 40 will be subjected to the ultrasonic energy.

In one exemplary embodiment, the sonication tank configured to containabout 20 gallons of sonication fluid for the treatment of allografttissues. In another exemplary embodiment, the sonication tank has aminimum volume of about 16 gallons and a maximum volume of about 26gallons.

In one exemplary embodiment, the sonication tank has a height rangingfrom about 33 to 49 inches to the top of the tank and about 37 to 53inches to the top of the cradle. In an alternative exemplary embodiment,the height of the sonication tank is about 33 inches to the top of thetank and about 37 inches to the top of the cradle.

In one exemplary embodiment, the rotary cleaning system can be used forthe treatment on a variety of different types of products. One exemplaryembodiment is the treatment of human cadaver allograft tissue.

Human cadaver allograft tissue includes, but not limited tomusculoskeletal, skin, osteoarticular and/or cardiovascular tissues.

In another exemplary embodiment, the rotary cleaning system can be usedto treat tissue engineered scaffolds, and polymeric or ceramic medicaldevice implants.

Sonication fluid 22 may comprise any of a variety of compositions, suchas water (an excellent medium for transmitting ultrasonic energy). Ofcourse any of a variety of other fluids may be used, such as phosphatebuffered saline or even a glycerol-based solution in order to helpmaintain the temperature of the sonication fluid. In addition, thesonication fluid may contain other fluids to alter the transfer ofultrasonic energy by increasing or decreasing depending on theapplication. The treatment canister may be made from any of a variety ofmaterials, including metal (particularly stainless steel), glass,polymeric materials (e.g., DELRIN® acetyl resin, PTFE, etc.).

Sonication tank 21 may include one or more heaters for maintaining thetemperature of sonication fluid 22. For example, the temperature ofsonication fluid 22 may be maintained at a temperature of between about20.degree. C. and about 70.degree. C., or between about 45.degree. C.and about 50.degree. C. It has been discovered that elevatedtemperatures facilitate the removal of protein and lipid constituents byincreasing the solubility of such contaminants. Elevated temperaturesalso greatly improve the log reductive capabilities of oxidizing andantimicrobial agents. In one exemplary embodiment for the treatment ofallograft tissues, the sonication fluid is maintained between about45.degree. C. to about 50.degree. C.

Sonication tank 21, and in particular ultrasonic transducers 30, may beconfigured to apply constant and/or pulsed ultrasonic energy withinsonication fluid 22 which in turn is transmitted into the interior oftreatment canister 40. For the application of pulsed ultrasonic energy,the frequency and/or power of the ultrasonic energy may be varied duringthe treatment of allografts. For example, in one embodiment, sonicationtank 21 is configured to apply ultrasonic energy at a frequency ofbetween about 40 kHz and about 170 kHz. In another embodiment, thefrequency of applied energy may be between about 72 and about 104 kHz,as such frequencies provide further improved cleaning of the protein andlipid constituents and liberation of viable microorganisms. In oneembodiment, the power output may be between about 100 watts/gallon andabout 550 watts/gallon. In another embodiment, power output is betweenabout 100 and about 300 watts/gallon. Intensities in excess of 550W/Gallon do provide additional microbial reductions, since such powerlevels result in the killing of more microbes. In one exemplaryembodiment, the ultrasonic energy is supplied by 3 to 4 generators witha combined power between 2000 and 2250 W. In another exemplaryembodiment for treatment of allograft products, the frequency of theapplied energy is 104 kHz. As one skilled in the art will appreciate,the frequency can be modified corresponding to the treatment of othermore sensitive or robust products. In one exemplary embodiment, thepower output is delivered to allograft tissues at about 100Watts/gallon. In another exemplary embodiment, the generators arelocated (e.g. under the sonication tank) such as to minimize exposure toany fluid leaks. Another embodiment could be that all electrical andpower components may be located adjacent to or in another area tofurther reduce the footprint of the system facilitate servicing andmaintenance procedures and to minimize any electrical hazards.

Sonication tank 21, in particular ultrasonic transducers 30 and anyheater(s) associated with the tank, may also be in electricalcommunication with a control system for the ultrasonic treatmentapparatus (e.g., controller 80) in order to control the application ofultrasonic energy and the temperature of the sonication fluid during thetreatment process. In one exemplary embodiment, an intensity measuringdevice is wired externally or internally to the sonication tank.

A treatment fluid is also provided in the interior of treatment canister40 in order to facilitate the application of ultrasonic energy to theallografts and optionally to perform other functions such as microbialreduction and cleaning (e.g., by killing or inactivating microbes).Ultrasonic energy from transducers 30 is transmitted through sonicationfluid 22, through the wall of treatment canister 40, and thereafterthrough the treatment fluid contained within treatment canister 40. Inthis manner, the ultrasonic energy is applied to the allografts so as toliberate microorganisms as well as residual proteins, lipids and tissueparticles from the allografts. Once the microorganisms are liberatedfrom their protective areas in the allografts the chemical agents in thetreatment fluid can more readily kill or inactivate the microbes out inthe open. The application of ultrasonic energy with rotation facilitatesthe liberation of microbes from the crevices of the tissue, andthereafter the liberated microbes are exposed to the treatment fluidsand higher temperatures so as to provide increased inactivation of themicrobes. Depending on frequency and power output, the ultrasonic energymay also inactivate microorganisms liberated from, or remaining presenton the allografts.

While the treatment fluid may comprise sterile water, applicants havealso discovered that the use of various other treatment fluids willfacilitate allograft cleaning and/or inactivation of microbialcontaminants, as further described herein. Applicants have alsodiscovered that the use of two or more different treatment fluids,particularly in a step-wise fashion, will further facilitate allografttreatment.

Any of a variety of treatment solutions may be used in the apparatus andmethods of the present invention, including sterile water. Othersuitable treatment solutions include detergent solutions, antimicrobialsolutions, strong bases, oxidizing agents, alcohols, and mixtures of theforegoing, including mixtures of one or more of the foregoing withsterile water. The order of treatment solutions may effect the cleaningand microbial inactivation. Detergent, enzymatic agents andantimicrobial detergents etc. may be used first to eliminate blood, bonemarrow and other organic matter. This eliminates strong reactions withoxidizing agents (e.g. hydrogen peroxide) that can create pressurewithin the sealed canisters and a potential hazard to the technicians.Typically, alcohol is used in the final step as a final microbialinactivation and drying agent. Sterile water can be used in betweensteps to further facilitate the removal of waste products and rinse thetissues of any residual chemical reagents that may impede finalmicrobial testing.

Suitable detergent solutions include lipases, proteolytic enzymes,lysozymes, and xyloansases. Antimicrobial detergents such as one or morepolymyxins (e.g., Polymyxin B may also be used). Detergents willliberate blood, bone marrow, microbes and other contaminants from theallografts. Some detergents, particularly antimicrobial detergents, alsoprovide antimicrobial properties (i.e., kill or inactivate microbes).

Suitable antimicrobial agents include sulfonamides, fluoroquinolones(e.g. Ciprofloxacin, Norfloxacin), penicillins, cephalosporins (e.g.Cefoxitin), tetracyclines (e.g. oxytetracyclines), aminoglycosides (e.g.streptomycin, gentamycin, neomycin), macrolide antibiotics (e.g.erythromycin, clindamycin), glycopeptide antibiotics (e.g. vancomycin,teicoplanin), lantibiotics (e.g. nisin), Bacitracin and Polymyxin. Suchmicrobial solutions may be used to kill or inactivate any bacteriapresent on the allografts or dislodged therefrom by the ultrasonicenergy and/or detergents.

Suitable oxidizing agents include hydrogen peroxide, ozone, pericidicacid, TAEDs (N,N,N′N′-Tetra Acetyl Ethylene Diamine), sodiumpercarbonate, sodium perborate, sodium polyacrylate, chloride andchlorinated compounds (e.g., chlorine dioxide, sodium hypochlorite,calcium hypochlorite), and potassium permanganate. The oxidizing agentsare very effective at killing or inactivating microbes liberated from orpresent on the allografts. They also play an important role in the“whitening” or bleaching of the allografts.

Alcohols used in treatment solutions may include isopropyl alcohol andethanol alcohol. Alcohols not only act as microbial reduction agents,they may also be used as dehydrating agents to remove moisture from theallografts (reduces the probability of microbial survival on allograftsduring storage). Strong bases, particularly strong inorganic bases suchas NaOH, may also be used for microbial destruction. Strong bases alsoprovide anti-prion capabilities.

As further discussed herein, the treatment solutions may be supplied totreatment canister 40 in a step-wise fashion while ultrasonic energy isapplied to the treatment canister. Alternately, the treatment canistermay be removed from the sonication tank in order to exchange thetreatment solution.

Treatment canister 40 in FIG. 1 is depicted as having a cylindricalshape. However, this shape is merely exemplary, as a variety of othershapes for treatment canister 40 may be utilized (as further discussedherein). Treatment canister 40 is also rotatably mounted withinsonication tank 21, such that treatment canister 40 can be rotated whileultrasonic energy is applied to the allografts contained therein.

In one exemplary embodiment, the treatment canister comprises acanister. The canister may be fabricated out of materials commonly knownto those skilled in the art. In another exemplary embodiment, thecanister comprises a stainless steel construction. The stainless steelconstruction is beneficial for ease of sanitization and sterilizationwhile allowing maximum ultrasonic energy transfer. As one skilled in theart will appreciate, the denser the material of construction, the moreultrasonic energy that is absorbed by the canister and not transferredto the products to be treated. Other exemplary materials of constructioninclude polymers, glass and other metals.

In one exemplary embodiment, the canister is configured in a cylindricalshape and comprises a baffle system to ensure or maximize constanttissue tumbling action. In an alternative embodiment, the canister isconfigured in various polygonal shapes. It is believed that thepolygonal shapes may obviate the need for an internal baffle system.

In another exemplary embodiment, the canister further comprises aninternal basket. The internal basket is configured to facilitate theaseptic loading and removal of product from the canister whileminimizing ultrasonic energy absorption. In one embodiment, the internalbasket is configured to facilitate the ability of fluid delivery to thecanister such that the basket rotates while the outer canister staysstationary and can be filled with fresh fluid while the expired fluid isexhausted. In one exemplary embodiment, the canister ranges from about2-8 inches in diameter and from about 4-18 inches in length. In anexemplary embodiment for treating machine grafts, the canister is about3 inches in diameter and about 4.5 inches in length. In anotherexemplary embodiment for treating cut tissue, the canister is about 6inches in diameter and about 13 inches in length. In yet anotherexemplary embodiment for treating soft tissue, the canister is aboutfive inches in diameter and about 6.5 inches in length.

Applicants have found that the combination of sonication with rotationof treatment canister 40 provides improved allograft cleaning andmicrobial inactivation. Rotation of treatment canister 40 duringsonication will not only increase the movement of treatment fluid withincanister 40, but will also cause rotation and tumbling of theallografts. The increased fluid movement also results in enhancedremoval of contaminants from the allografts, while the tumbling of theallografts will not only cause more contaminants to be liberated fromthe allografts but will also expose more surface area of the allograftsto the ultrasonic energy. Without rotation of the treatment canister,particularly when multiple allografts are positioned therein, Applicantsbelieve that some of the allografts (or portions thereof) are shieldedfrom the ultrasonic energy by other allografts. Applicants believe thatrotation of treatment canister 40 significantly reduces (or eliminates)this shielding effect and ensures that the entire surface of each of theallografts is exposed to the ultrasonic energy. As further describedherein, these effects may also be enhanced by causing treatment fluid toflow through or within treatment canister 40 during sonication.

By way of example, allografts to be treated are placed within treatmentcanister 40. Thereafter, a treatment fluid is supplied to treatmentcanister 40, such as by pouring a treatment fluid into treatmentcanister 40, and the canister is then sealed. The filled treatmentcanister 40 is then mounted within sonication tank 21 and ultrasonicenergy applied to the interior of sonication tank 21 while treatmentcanister 40 is rotated. Ultrasonic energy is applied in treatmentapparatus 20 for a period of time, and at frequency and power sufficientto dislodge contaminants (e.g., microorganisms, residual proteins,residual lipids, residual tissue particles, etc.) from the allograftand/or to inactivate microbial contaminants present on or liberated fromthe allografts.

In one exemplary embodiment, the volume of treatment fluid added to thecanister is dependent upon the mass of the tissue that is being treated.In one particular exemplary embodiment, the volume of the treatmentfluid is in a ratio of 1:2 of solution (ml) to tissue (g). In analternative exemplary embodiment, the volume of the treatment fluid isin a ratio of 1:5 of solution (ml) to tissue (g). In yet anotherexemplary embodiment, the volume of the treatment fluid is in a ratio of1:3 of solution (ml) to tissue (g). In one exemplary configuration, thevolume of treatment fluid ranges from about 200 ml to about 3200 ml andthe mass of tissue treated ranges from 400 g to about 6400 g percanister.

If a treatment protocol utilizing a plurality of different treatmentfluids is to be followed, treatment canister 40 may be removed fromsonication tank 21, and the first treatment fluid drained from canister40. Thereafter, the second treatment fluid may be supplied to treatmentcanister 40 (e.g., by pouring the treatment fluid into the canister) andthereafter the canister is sealed and inserted back into sonication tank21. Ultrasonic energy may thereafter be applied to treatment canister 40in the same manner as described previously. This process may be repeatedusing any number and variety of treatment fluids, according to thepredetermined protocol. If desired, after a treatment fluid is drainedfrom treatment canister 40, sterile water (or other fluid) may be addedto treatment canister 40 for purposed of rinsing or flushing theallografts in order to remove any additional contaminants liberated fromthe allografts and any remaining treatment solution. The sterile waterwash may then be discarded and the next treatment solution added totreatment canister 40 and the canister then placed back into sonicationtank 21.

It should also be pointed out that it may be desirable to simply replacea particular treatment fluid in canister 40 with fresh treatment fluidof the same composition. For example, some of the reagents used in thetreatment fluid may lose strength or otherwise become less effectiveover time (e.g., hydrogen peroxide). In addition, waste products mayaccumulate in the treatment fluid (e.g., residual tissue particulates,blood, proteins, lipids, killed or inactivated microbes liberated fromthe allografts, etc.). Therefore, a particular treatment fluid may bedrained from canister 40 in the manner described above and thereafterreplaced with more of the same treatment fluid composition. This willnot only replenish the particular treatment fluid in order to maintainthe maximum reactivity between the treatment fluid reagents and theallografts, but will also move waste products (e.g., blood, bone marrow,microbes liberated from the allografts, etc.) away from the allografts.In this manner, the allografts will not remain in a weakened and/orcontaminated treatment fluid.

It is also contemplated that treatment fluid within treatment canister40 may be drained and replaced with the same or a different treatmentfluid aseptically without opening the sealed treatment canister. Forexample, treatment canister 40 may be configured so as to include adrain or other fluid passageway which may be opened without unsealingthe entire treatment canister. As further described herein in connectionwith FIG. 6, the treatment canister may include, for example, a springloaded drain such that the treatment canister may be placed intoreceptacle and then pushed downwardly so as to open the spring-loadeddrain. This will result in fluid within the treatment canister beingdrained along with any waste products present therein. Thereafter, freshtreatment fluid (either the same composition as or a differentcomposition from the treatment drained from the canister) may beaseptically added to treatment canister 40 without opening the sealedcanister.

Alternatively and as depicted schematically in FIG. 1, the treatmentapparatus according to embodiments of the present invention may beconfigured to supply one or more treatment fluids to the interior oftreatment canister 40 after the canister is mounted in sonication tank21, thereby avoiding the need for a manual fluid exchange or otherwiserequiring that canister 40 be removed from the sonication tank. In fact,particularly when more than one treatment fluid is used for treatingallografts contained within treatment canister 40, an automated systemfor supplying treatment fluids to canister 40 may be provided.

In the embodiment of FIG. 1, a treatment fluid input line 71 providesfluid communication between one or more treatment fluid sources (e.g.,treatment fluid reservoir 60) and the interior of treatment canister 40.Similarly, a treatment fluid outlet line 80 may be provided in order toallow treatment fluid to be expelled from the interior of treatmentcanister 40.

A plurality of treatment fluid sources may be provided, as shown in FIG.1, such as treatment fluid reservoirs 60, 62, 64 and 66. These treatmentfluid reservoirs may contain any of a variety of treatment fluids (asdescribed previously) which are supplied to treatment canister 40 duringallograft processing. For example, treatment fluids such as enzymesolutions, antibiotic solutions, oxidizing agents, and alcohols may beprovided, along with one or more sources of water (e.g., sterile water).

As further detailed herein, some allograft treatment regiments includesupplying a variety of treatment fluids to the allografts duringsonication, with a sterile water flush of treatment canister 40 betweentreatment fluids. In such instances, a single source of sterile water(e.g., reservoir 66) may be provided, with water supplied to treatmentcanister 40 as needed. In such an embodiment, the sterile waterreservoir may be larger than the other treatment fluid reservoirs.

In some embodiments, it may generally be desired to supply heatedtreatment fluid to treatment canister 40, even if sonication tank 21 isheated. For example, treatment fluid reservoir 60, 62, 64 and 66 may beheated in order to maintain the treatment fluids at the desiredtemperature (e.g., between about 37.degree. C. and about 50.degree. C.,or even between about 45.degree. C. and about 50.degree. C.).Alternatively, or in addition thereto, one or more in-line heaters maybe provided between treatment fluid reservoirs and treatment canister40, before and/or after manifold 70, such as one or more heatexchangers.

It may also be desirable to filter sterilize one or more of thetreatment fluids prior to the treatment fluid being supplied totreatment canister 40. For example, one or more in-line filters may beprovided between the fluid treatment reservoirs and manifold 70, and/orone or more in-line filters between manifold 70 and treatment canister40.

As mentioned previously, the treatment process may be automated, atleast in part, such that treatment fluid is supplied to treatmentcanister 40 according to a predetermined schedule and/or without theneed for an operator to open treatment canister 40 to exchange onetreatment fluid for another. In the embodiment shown in FIG. 1, each ofthe treatment fluid reservoirs is in fluid communication with a manifold70 (or similar device) which in turn is in communication with fluidinlet line 71. Manifold 70 is configured to control the flow oftreatment fluid from the reservoirs (60, 62, 64 and 66) into theinterior of treatment canister 40. A controller 80, such as a PLC orother processing device, can be used to control manifold 70. As alsoshown in FIG. 1, pumps 61, 63, 65 and 67 may be provided for reservoirs60, 62, 64 and 66, respectively, in order to deliver treatment fluidfrom the reservoirs to manifold 70. Controller 80 may also be used tocontrol these pumps in order to regulate the delivery of treatment fluidto canister 40.

As treatment fluid is delivered to treatment canister 40, any treatmentfluid already within the canister will be expelled through fluid outletline 80. A pump 81 may be provided in order to facilitate removal oftreatment fluid from canister 40, and may be controlled by controller80. A valve 82 may also be provided on outlet line 80 in order to directexpelled treatment fluid to disposal or to a filtering device (e.g., afilter). In the latter case, the expelled treatment fluid is passedthrough the filter, and thereafter the filter analyzed for microbialcontamination. In particular, after the final treatment step has beencompleted, the expelled final treatment may be analyzed for microbialcontamination for contamination. In this manner, microbial contaminationof the allografts may be assessed following the treatment process. Thismethod of assessing microbial contamination may follow the methodsdescribed in U.S. patent application Ser. No. 10/976,078, filed Oct. 28,2004, which is incorporated herein by way of reference.

Treatment canister 40 may be rotatably mounted within sonication tank 21in a variety of manners, such as the exemplary embodiment shown in FIG.2. In the embodiment of FIG. 2, a motor 25 and associated pulley 26 aremounted outside of tank 21, as shown. A belt 27 extends around pulley 26and a portion of one end of the treatment canister 40. Canister 40 isrotatably mounted within tank 21 such that, as pulley 26 is rotated bymotor 25, treatment canister 40 will rotate within tank 21. Of coursethe treatment canister may be rotated by any of a variety of mechanisms,such as direct drive, internalized gearing, In one exemplary embodiment,the canister is placed at a angle in the treatment apparatus such thatthe angle is configured to allow the treatment fluid to drain from thecanister. This is an alternative embodiment to the utilization of pumpsand/or rotation. etc.

The treatment canister may have any of a variety of shapes andconfigurations, such as the cylindrical shape shown in FIGS. 1 and 2.Alternatively, the interior of the treatment canister may have anon-circular cross-sectional shape in a plane orthogonal to therotational axis of the canister. For example, FIG. 3 depicts a treatmentcanister 140 which has a triangular cross-sectional shape. Suchnon-circular cross-sectional shapes for the interior of the treatmentcanister will not only increase turbulent fluid flow within treatmentcanister 140, but will also enhance the tumbling action of the allograftwithin the treatment canister. Both of these actions are believed toresult in not only the dislodgment of additional contaminants from theallografts, but also increase the amount of surface area of theallografts exposed to the ultrasonic energy.

As an alternative, or in addition to a non-circular interior shape forthe treatment canister, one or more baffles or other structures may beprovided inside the treatment canister in order to increase the tumblingaction of the allografts. For example, FIG. 4 depicts a treatmentcanister 240 comprising a cylindrical housing having a baffle insert 245positioned therein. Insert 245 may have any of a variety of shapes andconfigurations designed to increase the tumbling of allografts withincanister 240 during rotation.

Alternatively, or in addition thereto, one or more grooves or ribs maybe provided on the inner surface of the treatment canister (e.g.,longitudinally-extending grooves or ribs similar to the rifling of a gunbarrel). Of course any of a variety of baffle structures or otherfeatures may be provided inside the treatment canister in order toincrease the tumbling action of the allografts, either by physicalcontact with the allografts or by structures which induce turbulenttreatment fluid movement within the canister. By way of further example,baffle inserts or other structures having a variety of shapes may beused, such as geometric shapes having defined edges (e.g., polygonalshapes such as octagons, pentagons, etc.). Such defined edges, whetherprovided on a baffle insert positioned in the treatment canister orformed in the interior surface of the treatment canister, will force theallografts to tumble as they contact the edges of the structures withinthe treatment canister, thus exposing additional surface area of theallografts to the ultrasonic energy.

Treatment canister 240 also includes a pair of end caps 241 which may besecurely attached to opposite ends of the treatment canister after oneor more allografts have been inserted into the treatment canister. Oneor more O-rings 242 may also be provided in order to seal the treatmentcanister, along with one or more latching mechanisms 243. Each end cap241 also includes a hollow shaft 246 which extends away from the end capand the treatment canister. Treatment fluid may be purged into orexpelled from the interior of the treatment canister 240 through thehollow shafts 246.

FIG. 5 depicts an alternative embodiment in which treatment canister 340is foraminous. In this embodiment, treatment fluid is supplied tosonication tank 321 and also acts as the sonication fluid duringtreatment. Because treatment canister 340 is foraminous, the treatmentfluid will pass into the interior of treatment canister 340 and contactthe allografts contained therein. Treatment canister 340 is mountedwithin sonication tank 321 such that it may be rotated therein duringtreatment, however, the mechanism for rotating canister 340 has beenomitted from FIG. 5 for purposes of clarity.

Treatment fluid may be provided in sonication tank 321 manually, such asby opening lid 331 and pouring in the treatment fluid(s) according to,for example, a predetermined protocol. Alternatively, an automatedtreatment fluid supply system may be provided. For example, a pluralityof treatment fluid reservoirs (360, 362, 364 and 366) may be providedalong with corresponding pumps (361, 363, 365 and 367, respectively). Amanifold 370 and controller 380 may also be provided in order to supplythe proper treatment fluid to sonication tank 321 at the appropriatetime according to a predetermined treatment protocol. Fluid supply line371 is in fluid communication with a fluid inlet 328 on the sonicationtank 321. Similarly, a fluid outlet 329 is provided for treatment fluidexpelled from sonication tank 321. The interior of treatment canister340 may be cylindrical, as shown. Alternatively, any of the non-circularcross-sectional shapes described previously may be used.

FIG. 6 is a schematic illustration of yet another embodiment of anultrasonic treatment apparatus 420 according to the present invention.In the embodiment of FIG. 6, system 420 is configured such that a pairof treatment canisters 440A and 440B may be simultaneously processedwithin sonication tank 421. In this manner allografts from two differentdonors may be processed simultaneously. In the embodiment of FIG. 6, thetreatment canisters 440A and 44B are sealed with respect to the interiorof sonication tank 421 and are configured to receive one or moreallografts therein. Sonication tank 421 is configured to receive asonication fluid, in the manner previously described. In one exemplaryembodiment, the treatment apparatus comprises up to four (4) treatmentcanisters for simultaneous processing. As such, four (4) donor productscan be treated in one processing session. In an alternative embodiment,the treatment apparatus can be configured to contain more than fourtreatment canisters.

Although not shown in FIG. 6, sonication tank 421 may once again includeone or more heaters in order to maintain the desired temperature withinsonication tank 421. The embodiment of FIG. 6 also includes a mechanismfor cooling the sonication fluid in order to further maintain thedesired temperature. Particularly, the system of FIG. 6 includes acooling bath 431 located directly beneath sonication tank 421. Althoughnot visible in FIG. 6, sonication tank 421 includes one or more drainsin the bottom thereof which allows sonication fluid to flow from thebottom of tank 421 into cooling bath 431. A recirculating chiller/pumpdevice 430 is also provided, and is in fluid communication with bothcooling bath 431 and sonication tank 421, as shown. During use,sonication fluid emptying from sonication tank 421 into cooling bath 431is chilled and returned to sonication tank 421 by recirculationchiller/pump 430. Of course, a suitable control system may be providedin order to regulate the operation of recirculating chiller/pump 430 inorder to maintain the desired temperature within sonication tank 421.Since the ultrasonic transducers within or associated with sonicationtank 421 will cause the temperature of the sonication fluid to rise, theuse of recirculating chiller/pump 430 will further facilitate thecontrol of the temperature of the sonication fluid during treatment. Thesystem shown in FIG. 6 also includes a power supply 480 which willprovide the necessary power for the various components of treatmentsystem 420.

As also shown in FIG. 6, treatment canisters 440A and 440B are rotatablymounted to a carriage which may be lowered into sonication tank 421. Inparticular, the carriage includes a motor 425 which may be configured tonot only cause the desired rotation of the treatment canisters 440A and440B, but also to lower and raise the treatment canisters intosonication tank 421. Alternatively, raising and lowering of thetreatment canisters may be done manually, or even under the automaticcontrol of a suitable control system. Pulleys 426 and belt 427 may beused to translate rotation of the motor shaft to the treatment canistersin order to effect rotation of treatment canisters 440A and 440B insonication tank 421. Of course FIG. 6 merely depicts one possible mannerin which the treatment canisters may be rotated within sonication tank421 during the treatment process.

In one exemplary embodiment, the rotation of the treatment canisterscomprises an indirect belt drive system with a rotation speed of about10 revolutions per minute. As one skilled in the art will appreciate,various rotation drive systems may be utilized in the present invention.Alternative rotation drive systems include, but are not limited todirect drive systems (i.e., with gears or solid drive systems) having avariable system with rotation speeds of between 0.1 and 60 revolutionsper minute. In one exemplary embodiment for treatment of allografttissues, the rotation speed ranges from 8 to about 12 revolutions perminute. As will be appreciated by one skilled in the art, a fixed speedor variable speed motor can be utilized for the rotation drive system.In one exemplary embodiment, the rotation drive system comprises a fixedspeed motor.

Treatment system 420 in FIG. 6 is also configured to facilitate theexchange of treatment fluids, particularly exchanging treatment fluidswithout opening the treatment canister. In addition, system 420 is alsoconfigured to not only process allografts from two different donors atthe same time (using first and second treatment canisters 440A and440B), but also to perform such processing without risk ofcross-contamination between the tissues of different donors. Inparticular and as shown in FIG. 6, treatment system 420 includes adrainage cradle 432 and a filling cradle 433. A drainage line 434 isprovided in fluid communication with drainage cradle 432 such thattreatment fluid and waste materials removed from a treatment canisterplaced into drainage cradle 432 will be drained through drainage hose434 (for discard or collection and subsequent analysis).

In the embodiment shown in FIG. 6, treatment system 420 is configured todeliver three different treatment fluids to the treatment canisters forallograft processing. Of course, any number of treatment fluids may beused and the treatment system configured accordingly. In the embodimentshown, fluid conduits 461, 463 and 465 are provided in fluidcommunication with one or more storage tanks for the treatment fluids tobe used. These conduits deliver the appropriate treatment fluid totreatment fluid delivery devices 460, 462 and 464, respectively. Thesedelivery devices may simply comprise a secondary storage tank from whichthe treatment fluid is delivered (e.g., by gravity feed) to manifold 470for transfer to a treatment canister via fill conduit 471 (e.g., ahose). Alternatively, fluid delivery devices 460, 462 and 464 may eachinclude a fluid pump, a heat exchanger, and/or a filtration system forthe corresponding fluid delivered thereto.

Each of the treatment fluid delivery devices is in fluid communicationwith a manifold 470, which in turn is in communication with treatmentfill conduit 471. A waste return conduit 467 is also provided in fluidcommunication with manifold 470, and may be used to drain excesstreatment fluid from manifold 470, as needed. In addition, or as analternative, waste return conduit 467 may be used to provide sterilizein-place capabilities (e.g., for sterilizing the interior of a treatmentcanister, manifold 470 or any of the other fluid delivery componentsdepicted in FIG. 6). In the particular embodiment shown in FIG. 6, fluidconduit 461 may be in fluid communication with a source of hydrogenperoxide, conduit 463 in fluid communication with a source of sterilizedwater, and fluid conduit 465 in fluid communication with a source ofisopropyl alcohol.

In one exemplary embodiment, the treatment apparatus is sanitized inplace, wherein the apparatus is sanitized using common methods known tothese skilled in the art such as flushing the treatment canisters andbaskets with heated water (approximately 70.degree. C. for 20 minutes)and/or flushing with chemical sanitization agents.

In order to use treatment system 420 of FIG. 6, allografts to be treatedare first placed within one of the treatment canisters 440A or 440B. Theends of the treatment canister (e.g., the treatment canister depicted inFIG. 4) are then sealed and the treatment canister is placed into fillcradle 433. The end of conduit 471 is then connected to an inletprovided on one end of the treatment canister. Treatment fluid is thenpurged into the treatment canister through conduit 471. Thereafter, thetreatment canister is loaded into the carriage, as shown. The othertreatment canister may, if desired, be similarly filled with allograftsto be treated, as well as the first treatment fluid to be used duringallograft treatment. Thereafter, the carriage is lowered into sonicationtank 421 and ultrasonic energy applied to the treatment canisters whilethe treatment canisters are rotated.

After a predetermined amount of time has passed, the carriage is raisedout of sonication tank 421 and one of the treatment canisters is removedfrom the carriage and placed into drainage cradle 432. At least one endof the treatment canister may be configured so as to include aspring-loaded drain. For example, the spring-loaded drain may beconfigured such that when the treatment canister is inserted intodrainage cradle 432 and pressed downwardly, a structure within drainagecradle 432 will act to open the spring-loaded drain. In this manner,used treatment fluid as well as waste materials will be drained from thetreatment canister through drainage conduit 434 without the operatoropening the treatment canister. Once drained, the treatment canister isthen placed into fill cradle 433 and fill conduit 471 is connectedthereto. The treatment canister may then be filled with a secondtreatment fluid (which may be the same or different than the firsttreatment fluid) or filled with a rinse solution which is thereafterdrained from the treatment canister using drainage cradle 432.

It is also contemplated that rinsing or flushing of the interior of thetreatment canister may be accomplished while the treatment canister ispositioned within drainage cradle 432 (e.g., by delivering a rinse fluidsuch as sterilized water to the treatment canister via fill conduit 471while simultaneously or periodically opening the spring-loaded drainprovided in the end of the treatment canister). Rinsing and/or flushingof the interior of the treatment canister may be repeated, as desired.Thereafter, the treatment canister is filled with the predeterminedtreatment fluid according to the predetermined processing steps usingfill cradle 433. The treatment canister is returned to the carriage andlowered back into sonication tank 421 for additional treatment. Thisentire process may be repeated as many times as desired, particularlyaccording to predetermined processing steps.

Another embodiment of an ultrasonic treatment apparatus 720 isillustrated in FIG. 7 according to the present invention. In thisembodiment, the ultrasonic treatment apparatus 720 is configured suchthat up to four canisters 725 may be simultaneously processed within thesonication tank 730. In this manner, allografts from one to fourdifferent donors may be processed simultaneously. In the presentembodiment, the apparatus 720 further comprises an operator controlinterface 740. The operator control interface 740 is configured suchthat a user can enter desired treatment parameters for the apparatus.

A variety of treatment protocols may be used, regardless of whichtreatment apparatus is employed. One advantage provided by embodimentsof the present invention is that the processing of allografts frombeginning to end can be performed in separate rooms or facilities havingdifferent sterility standards without jeopardizing allograft sterility.

For example, recovered tissue may first be processed in a recovery room.This processing may include debridement, and the removal of grossmusculature, connective tissues and blood elements. The intact tissuesare then cut into allograft products. Thereafter, the allografts may beloaded into a treatment canister (e.g., treatment canister 40 from FIG.1). In some embodiments, the treatment canister may be sealed at thistime.

The allograft-loaded treatment canister is then taken to a secondfacility or room, such as an allograft processing room, for treatmentaccording to the present invention. For example, an allograft-loadedtreatment canister may be placed into the sonication tank. Thereafter,the first treatment fluid is automatically pumped into the canisterwithout opening the canister or exposing the allografts thus preservingthe integrity of the cleaned allografts as well as minimizingenvironmental cross-contamination. The allografts are subjected tosonication and rotation according to a predetermined schedule.Periodically, the treatment fluid in the treatment canister may beexchanged, such as by pumping additional treatment fluid (either thesame or different from that already in the canister) while allowing thetreatment fluid already in the canister to escape (and optionallypumping out the treatment fluid already in the canister).

Following treatment, the allografts, still contained within the sealedtreatment canister, may be taken to a third facility, biological safetycabinet or room, such as a packaging room. Here the allografts may bevisually inspected, measured and aseptically packaged for subsequentterminal sterilization or delivery to practitioners for use.

Referring now to FIGS. 8 and 9, and in another embodiment, a deliverysystem 800, 805 may be provided to aseptically transfer fluids to andfrom multiple canisters 810A, 810B, 810C, 810D. System 800, 805 may alsobe configured to prevent potential contamination between the canisters810A, 810A, 810C, 810D and from the outside environment into canisters810A, 810B, 810C, 810D. Although not limited to this configuration,system portion 800 includes canisters 800A, 800B, 800C, 800D in a bathand system portion 805 includes reagent tanks 815 on scales. Otherconfigurations may be utilized. System 800, 805 may be used with therotary cleaning system for cleansing of allografts, but may beincorporated into other systems.

In one embodiment, system 800, 805 may deliver sterile fluidsaseptically to sealed canisters 810A, 810B, 810C, 810D. In anembodiment, the sterile fluids are delivered from reagent tanks 815.(See FIG. 9). System 800, 805 may deliver fluids aseptically from sealedcanisters 810A, 810B, 810C, 810D to a drain system 820.

In an embodiment, system 800 has vents (described below) to allow forout-gassing of materials and expansion of air due to heating duringprocessing while maintaining a sterile barrier between canisters 810A,810B, 810C, 810D and the outside environment. This prevents potentialenvironmental contamination from entering sealed canisters 810A, 810B,810C, 810D. For the rotary cleaning system, canisters 810A, 810B, 810C,810D may be submerged in a common water bath 825. With the sealedsystem, potential contamination is also prevented from being transferredfrom one canister 810A, 810B, 810C, 810D to another canister 810A, 810B,810C, 810D. This also prevents mixing of materials from one canister810A, 810B, 810C, 810D to another canister 810A, 810B, 810C, 810D, aswell as with the common water bath.

Reagents may be delivered in prescribed amounts with system 800, 805.For the rotary cleaning system configuration reagents may be deliveredfor each processing step in a ratio of 1.8 to 2.2 milliliters of reagentfor each gram of tissue or any other prescribed ratio.

System 800, 805 and sealed canisters 810A, 810B, 810C and 810D may bedetermined to be leak tight prior to the beginning of processing. Thisensures that material/equipment defects or assembly error have notcreated a leak path. This is important with the rotary cleaning systemsince the sealed canister 810A, 810B, 810C, 810D and some of the reagentdelivery system 800 is submerged in water bath 825, which may transmitenvironmental contamination or contamination from one canister 810A,810B, 810C, 810D to another canister 810A, 810B, 810C, 810D if there areleaks.

Assemblies forming canisters 810A, 810B, 810C, 810D may be sealed to theoutside environment utilizing o-rings 828 or other seals 828 between allmetal-metal interfaces that are not sealed using a weld. A static sealconfiguration may be used for parts that do not move relative to oneanother. A dynamic seal configuration may be used for parts that rotaterelative to one another.

Sterilized tubing 830 is utilized to move reagents between components.Standard barb fitting configurations 835 are used and tubing 830 is slidover these barbs 835. To ensure a 360° seal between barb 835 and tubing830, as well as to ensure the tubing 830 does not move relative to barb835 (e.g. due to vibration from the process) a BarbLock® tubing retainermay be utilized. Tubing 830 that could potentially be contaminated bymaterials in one of the canisters 810A, 810B, 810C, 810D is changed eachtime canister 810A, 810B, 810C, 810D, or tissue material, is changed.

Pinch valves 840 may be utilized to isolate various fluid containingcomponents from one another. By utilizing a pinch valve 840 there is noneed to sterilize any of the valve components forming one of the pinchvalves 840 since the valve only contacts the outside of the tubing 830does not contact sterile reagents.

Leak testing is performed prior to the start of processing session toensure that seals 828 are intact and tubing 830 is assembled properly.An initial leak test may be performed up to a reagent delivery manifold845. A pressure transducer 850 (see FIG. 8) measures manifold pressure,checking for the proper range of pressure and looking for pressuredecays which would indicate a leak. Additional leak tests performed mayinclude reagent delivery manifold 845 and the canisters 810A, 810B,810C, 810D being used. Testing of each canister 810A, 810B, 810C, 810Dis done individually. A leak in any system requires correction beforethe delivery process can begin.

The sequence of opening valves 840 ensures that potential contaminationis not transmitted from one canister 810A, 810B, 810C, 810D to another.Upstream valves 840A (see FIG. 8) are opened first (prior to downstreamvalves 840B) to ensure positive pressure and flow both into and out ofeach canister 810A, 810B, 810C, 810D. When upstream valves 840A areclosed, downstream valves 840B are closed first for the same reason.

The materials used in the process may outgas thereby producing pressurein canister 810A, 810B, 810C, 810D that could lead to excessivestructural loading on system 800. Changes in the temperature of canister810A, 810B, 810C, 810D may also create a pressure or a vacuum. Toaddress this, a vent port 850 is included on canister 810A, 810B, 810C,810D. (See FIGS. 8, 11, and 12A-12D.) A vent tube 830A is attached tothis port 850 and then attached to an “alcohol bubbler” 855. (See FIG.10.) This concept is similar to bubblers used in the wine and beermaking process. A bottle 860, which may include a lid 862, may be filledto a prescribed level with isopropyl alcohol 865. A dip tube 870connects to the vent tubing 830A and the open end is held below thelevel of the alcohol 865. A one way valve 875 is located between venttubing 830 and dip tube 870 which prevents flow from the alcohol intothe vent tubing 830. Out gassing or pressure created by a temperatureincrease is released into isopropyl alcohol 865 (i.e. “bubbler”).Isopropyl alcohol 865 acts as a microbial barrier between the externalenvironment and vent tubing 830 leading back to canister 810A, 810B,810C, 810D.

Referring again to FIG. 9, delivery of reagents 880 is accomplishedusing sterile nitrogen 885 to pressurize reagent tanks 815A, 815B, 815C,815D. A nitrogen inlet fitting 895 at the top of the tank 815A, 8158,815C, 815D allows reagent 880 to be pressurized from above. A reagentoutlet fitting 900 is attached to a dip tube 905 that allows reagentfluid 880 to be drawn from the bottom of tank 815A, 815B, 815C, 815D.(See FIG. 9.)

Canisters 810A, 810B, 810C, 810D each have three fittings each of whichis a different size to mistake-proof assembly of the correspondingtubing 830 (see FIGS. 9, 11 and 12A-12D). Inlet fitting 910 is placedabove the air headspace in canister 810A, 810B, 810C, 810D and allowsfor delivery of reagent 880 into canister 810A, 810B, 810C, 810D. Drainfitting 915 is placed at the bottom of canister 810A, 810B, 810C, 810Dand allows for reagent 880 to be purged out of canister 810A, 810B,810C, 810D and into a drain system. Vent fitting 850 is placed at thetop of canister 810A, 810B, 810C, 810D and allows for pressure incanister 810A, 810B, 810C, 810D to remain at ambient pressure asdescribed above.

Reagents 880 are removed from canisters 810A, 810B, 810C, 810D usingsterile nitrogen 925 to pressurize canisters 810A, 810B, 810C, 810D.Vent pinch valve (not shown) 840C is closed and the drain valve (notshown) 840D is opened to maximize the amount of reagent 880 removed fromthe canister 810A, 810B, 810C, 810D.

The reagent tanks 815A, 815B, 815C, 815D reside on weigh scales 930A,930B, 930C, 930D. (See FIG. 9.) An initial tare weight is recorded andthen a set amount of reagent 880 is delivered based upon the weight ofthe donor tissue in the canister 810A, 810B, 810C, 810D. The fastestflow may be determined empirically and used for the initial delivery ofreagent 880. The change in scale weight is determined and then comparedto the required weight. If additional reagent 880 is required,additional fills are performed until the weight is within therequirements.

A nitrogen manifold 935 is utilized to distribute nitrogen 940 atpressure from one main source to each of the reagent tanks 815A, 815B,815C, 815D and to a reagent delivery manifold 845 for purgeapplications. (See FIG. 9.)

Reagents 880 are distributed to up to four canisters 810A, 810B, 810C,810D using a common manifold 845 (see FIG. 8). Manifold 845 has an inlet945 for each of reagents 880 and for nitrogen 885 with pinch valves 840Aon the tubing 830 just prior to manifold 845. Manifold 845 has fouroutlets 950 for each of the four potential canisters 810A, 810B, 810C,810D. Each outlet 950 has a pinch valve 840B on tubing 830 immediatelyafter the manifold outlet 950. By limiting the size of inlet/outlet945/950 volumes, and the volume of the manifold 845 itself, mixing ofreagents 880 is minimized. In addition, the purging of reagents 880 inbetween uses with nitrogen 885, limits the mixing of residual reagents.

Interface and Calculations:

A Human Machine Interface (HMI) may be utilized for an operator to inputkey parameters needed for proper delivery of reagents. The operator mayidentify the amount of tissue and, the tissue type for each canister.This information may be stored in a Programmable Logic Controller (PLC)for determining the reagent weight requirements and for the proper“recipe” for the tissue being processed.

All critical parameters, such as reagent amounts and exposure times, maybe transferred for the PLC to a separate computer system. In-housesoftware may be configured to receive this information and place thisinformation in a non-alterable format. This information may be printedat the end of the processing session and is backed-up on server.

EXAMPLES

One process for treating cut allografts (i.e., cortical/cancellous,cancellous, soaking grafts and machine grafts) according to anembodiment of the present invention is as follows:

TABLE-US-00001 Treatment Time Temp Rotation Parameter (rpm) Step 1: DrySpin Centrifugation @ 3 minutes N/A 1460 RCF Step 2:Bacitracin/Polymyxin B* 30 minutes 45-50.degree. C. ˜10 RPM/104 kHz (100W/Gal) Step 3: Static Sterile Water Rinse ˜0.5 minutes 22-50.degree. C.N/A Step 4: 3% Hydrogen Peroxide 60 minutes 45-50.degree. C. ˜10 RPM/104kHz (100 W/Gal) Step 5: Static Sterile Water Rinse ˜0.5 minutes22-50.degree. C. N/A Step 6: Bacitracin/Polymyxin B 60 minutes45-50.degree. C. ˜10 RPM/104 kHz (100 W/Gal) Step 7: Static SterileWater Rinse ˜0.5 minutes 22-50.degree. C. N/A Step 8: 3% HydrogenPeroxide 60 minutes 45-50.degree. C. ˜10 RPM/104 kHz (100 W/Gal) Step 9:Static Sterile Water Rinse ˜0.5 minutes 22-50.degree. C. N/A Step 10:70% Isopropyl Alcohol 30 minutes 45-50.degree. C. ˜10 RPM/104 kHz (100W/Gal) Step 11: Static Sterile Water Rinse ˜0.5 minutes 22-50.degree. C.N/A Step 12: Static Sterile Water Rinse ˜0.5 minutes 22-50.degree. C.Total Processing Time 242.5 min. Net Log Reduction >4 logs *50 U/mLBacitracin and 500 U/mL Polymyxin B

One process for treating soft tissue (i.e. ligament and tendon tissueswith or without bone blocks) according to an embodiment of the presentinvention is as follows:

TABLE-US-00002 Treatment Time Temp Rotation Parameter (rpm) Step 1: DrySpin Centrifugation @ 3 minutes N/A N/A 1460 RCF Step 2: Polymyxin B(1000 U/mL) 30 minutes 45-50.degree. C. ˜10 RPM/104 kHz (100 W/Gal) Step3: Static Sterile Water Rinse ˜0.5 minutes 22-50.degree. C. N/A Step 4:70% Isopropyl Alcohol 3 minutes 45-50.degree. C. ˜10 Step 5: StaticSterile Water Rinse ˜0.5 minutes 22-50.degree. C. N/A Step 6:Bacitracin/Polymyxin B* 30-35 minutes 45-50.degree. C. ˜10 RPM/104 kHz(100 W/Gal) Step 7: Static Sterile Water Rinse ˜0.5 minutes22-50.degree. C. N/A Step 8: Static Sterile Water Rinse ˜0.5 minutes22-50.degree. C. N/A Total Processing Time 67.5 min. Net Log Reduction˜3 logs

The specific illustrations and embodiments described herein areexemplary only in nature and are not intended to be limiting of theinvention defined by the claims. Further embodiments and examples willbe apparent to one of ordinary skill in the art in view of thisspecification and are within the scope of the claimed invention.

1. A method of delivering sterile fluids aseptically to a sealedcanister, the method comprising: providing a canister having an inlet, avent, and a drain outlet; selectively providing a selected one of aplurality of reagents to the canister from a reagent manifold in sealedfluid communication with the inlet of the canister; allowing excessgasses to leave the canister with a bubbler in sealed fluidcommunication with the vent of the canister; and selectively purging theselected one of the plurality of reagents from the canister through thedrain outlet into a fluid communicator to a drain.
 2. A method inaccordance with claim 1, further comprising delivering the sterilefluids aseptically from a storage/reagent tank through the reagentmanifold to the canister.
 3. A method in accordance with claim 1,wherein allowing the excess gasses to leave the canister providesout-gassing of materials and expansion of air due to heating duringprocessing while maintaining a sterile barrier between the canister andan outside environment.
 4. A method in accordance with claim 1, furthercomprising submerging a plurality of canisters in a common water bath,and preventing potential environmental contamination from entering thecanister.
 5. A method in accordance with claim 1, further comprisingsubmerging a plurality of canisters in a common water bath, and furthercomprising preventing potential contamination from being transferredfrom one canister to another canister.
 6. A method in accordance withclaim 1, further comprising submerging a plurality of canisters in acommon water bath, and further comprising preventing of mixing ofmaterials from one canister to another.
 7. A method in accordance withclaim 1, further comprising deliver reagents in prescribed amounts.
 8. Amethod in accordance with claim 1, further comprising ensuring that thesealed canister, reagent manifold, and bubbler in sealed fluidcommunication with one another are leak tight prior to the beginning ofprocessing.